: Recombinant AAV has several characteristics that underscore its potential as a gene therapy vector which include its ability to integrate into the genome of non-dividing cells, the lack of immune response since all viral genes can be deleted, and the fact that rAAV can be concentrated to high titers. In the previous funding period several strategies were used to evaluate and improve rAAV as a tool for gene therapy in Cystic Fibrosis (CF). In this proposal the specific aims will attempt to delineate cellular pathways underlying rAAV transduction in the airway and then to exploit this basic understanding to enhance its utility for gene therapy in CF. Four specific aims are described to answer the following: 1) what are the mechanisms of rAAV entry and trafficking to the nucleus in polarized airway epithelial; 2) what factors control the processes of AAV circular intermediate formation during latent phase infection; 3) are circular AAV genomes preintegration intermediates, and what structural features influence the efficiency of long term persistence; and 4) will concatamers of rAAV circular genomes prove to be useful delivery systems of full length CFTR? The proposal will focus on the understanding of the current barriers to rAAV transduction in the airway, to develop approaches to maximize gene transfer.